Cell culture method

ABSTRACT

A method for culturing animal cells in vitro in a monolayer by supporting the cells on the surface of a solid porous matrix and contacting the cells so supported with a liquid medium in the form of a foam. As the foam comes into contact with the matrix it breaks down and flows over the surface of the cells as a film, leaving gas in the interstitial spaces.

This application is a continuation-in-part of my application Ser. No.686,097 filed May 13, 1976 and now abandoned.

The invention relates to an improved method for the monolayer culture ofanimal cells in vitro and an apparatus to operate the method.

There are two chief methods for culturing discrete cells ormicro-organisms in vitro: firstly as a suspension in a stirred tankreactor and secondly as a monolayer attached to a supporting surface.Generally, a suspension culture is only suitable for established celllines, cells of the lymphocytic type including normal lymphocytes, tumorcells such as Burkitt lymphoma cells and leukaemic myoblasts. Cellsforming sheets or strands such as fibroblasts and epithelial cellsunless transformed require a solid support upon which to grow and it iswith the latter method of culturing that the present invention isconcerned.

Laboratory scale monolayer culture is a simple process in which cellsare supported and grown on the inside walls of a static or rotatingbottle. It is well known that for mass monolayer culture, cells must beprovided with a large surface area. The rotating bottle has been adaptedfor large scale production by linking together a number of culturevessels so that culture medium may be continuously pumped through aseries of bottles. However, methods employing this type of apparatus arelabour intensive, since subculturing is often required, and theapparatus itself is costly, large and mechanically complex.

An apparatus for providing a large surface within a single culturevessel is described by W. Wohler et. al. in the Journal of ExperimentalCell Research Volume 92 1972, page 571, and is the subject of WestGerman Offenlegungsschrift No. 2,300,567. The apparatus consists of aconical flask containing a high surface area matrix such as a mass ofglass beads. The cells are applied to the matrix which is submerged inthe culture medium and the medium is continuously replenished.

The disadvantages of this submerged culture technique are:

(1) that the apparatus required to maintain the medium characteristicsis complex; and

(2) that due to differences in concentration in the medium covering thecells it does not give in general as good growth conditions as therotating bottle, in which cells are covered by a thin film of growthmedium.

The present invention is based on the discovery of a novel method ofsupplying a thin film of nutrient medium to discrete cells in amonolayer.

According to this invention there is provided a method for culturinganimal cells wherein a monolayer of the cells supported on the surfaceof a solid porous matrix, is contacted wtih a film of liquid nutrientmedium derived from the breakdown of a foam, which is generated fromnutrient medium and a gas.

In the method of this invention, a monolayer of cells is attached to atleast part of the solid porous matrix, for example by steepin the matrixin a suspension of the cells in a liquid medium, allowing the cells tosettle on the matrix and subsequently draining the suspension medium.Nutrient medium in the form of a foam is then supplied to the matrix ata sufficiently fast rate to prevent the matrix from drying out, whilstthe cells are maintained at a suitable physiological temperature (i.e.35°-38° C.). As the foam comes into contact with the matrix, it breaksdown and flows as a thin film throughout the matrix, over the surface ofthe cells, while leaving the gas in the interstitial spaces. The mediumflowing from the matrix may be run to waste or, where suitable,recycled. The cells are cultured until the matrix is covered with asufficiently large monolayer of cells. The cells are then washed toremove nutrient medium optionally using a film derived from a foamedwash medium. The cells are then removed by any standard method, forexample by trypsinization optionally with gentle agitation of thematrix, and then collected.

This method is suitable for culturing those cells derived from animals,including mammals, birds, amphibia, fish and insects, which grow insheets in vitro. Examples of such cells are those derived fromEpithelial tissue, Connective tissue, Muscle tissue, Nervous tissue andLymph tissue. In particular, the method is applicable to culturing skinand muscle fibroblasts derived from mammals including man.

The matrix used in this invention is porous. The interstitial spaces ofthe porous matrix must be sufficiently large, (a) to accommodate thecell monolayers; (b) to allow the culture medium to flow as a thin filmacross the monolayer surface, while retaining gas in spaces; and (c) toallow the removal of the cells.

The surface of the porous matrix of this invention must be capable ofadhering the cells to be cultured and capable of releasing the cellswhen the culture process is complete. It is well known that animalscells in particular adhere well to surfaces which carry high densitiesof sodium ions. They therefore adhere to materials which tend to acquirea negative charge and thus bind sodium ions. The matrix may be made froma material to which cells adhere or be made up from an inert support andcoated with such a material. Suitable materials include plastics,materials such as nylon, polycarbonate, polystyrene, epoxyresins,silicone rubber, cellulose acetate, cellulose nitrate, cellophane andP.T.F.E., polyethylene terephthalate, polyformaldehyde, fluorinatedethylenepropylene co-polymer, polyphenylene oxide, polypropylene mica,carbon, collagen, insoluble inert metal oxides, phosphates, silicates orcarbides, silicon carbide, inert metals such as stainless steel,aluminium, titanium or palladium, or ceramics or glass. We have foundthat different cell types adhere to the matrices with differing degreesof tenacity. For exaple some cell lines adhere so well to some of theabove types of support that it becomes difficult to remove the cellswhen harvesting is required. Thus it is sometimes necessary to modifythe characteristics of the surface by applying a coating of a materialless adhesive to cells, which eases their removal. Coating materialswhich we have found render cells more readily removable from matrixsurfaces are polyfluorinated hydrocarbons such aspolytetrafluoroethylene, or silicones such aspolymethylhydrogensiloxane. On the other hand a surface of low adhesivecapacity may be altered to give better adhesion by the application of asuitable coating.

Thus the particular surface coating which is employed will depend uponthe type of cells to be cultured and whether harvesting from the matrixis required. A suitable coating or combination of coating for anyparticular application may be determined empirically.

Materials which we have found in particular to be suitable for theformation of matrices for use in this invention include polycarbonate,nylon 6, nylon 11, nylon 12, glass, polyformaldehyde, polypropylene and2,6-dimethylphenyleneoxide. Coated matrices which we have in particularfound to be suitable for the formation of matrices for use in thisinvention include polycarbonate coated with polytetrafluoroethylene,silicone, polymethylhydrogensiloxane; glass coated with silicone,polytetrafluoroethylene or stearic acid; or polyethyleneterephthalate,nylon 6, nylon 11 and nylon 12, each coated withpolytetrafluoroethylene.

In any specific application of this method the particular material ofchoice may be determined by incidental factors such as the method bywhich the matrix is to be sterilized.

The matrix may be made from one or more macroreticular solid pieces.Such matrices may be for example, a solid foam formed in a materialcapable of supporting cells, e.g. a plastics material such as nylon,polycarbonate, polystyrene or an inorganic material such as silica gel.

Alternatively the matrix may be in the form of a plug of fibrousmaterials such as a metal wire wool, a plastics wool or glass wool. Inthese circumstances the material would be loosely packed in order toprevent any coalescence of the thin film flow into liquid dropssubmerging part of the matrix. A further type of matrix may take theform of a regular lattice of threads, such as net, made up from forexample plastics, metallic, or glass thread.

In yet another embodiment the matrix may consist of a mass of regular orirregular solid or hollow granules made from or coated with the types ofmaterials previously discussed. The shape and size of the granules isnot critical. Regular shapes such as spheres or cylinders are preferredto irregular shapes, since it has been found that nylon cylinders weremore suitable than the moulding chips from which they were prepared.Spheres are more suitable than cylinders since for a given volume,spheres have a higher surface area.

For spheres, suitable diameters are in the range 1-10mm, preferably inthe range 2-4mm. The diameters of cylinders are suitably in the range1-10mm, preferably 3-5mm; and the length of cylinders may be from1-10mm, preferably 3-5mm. Particularly suitable cylinders have adiameter of 4mm and a length of 4mm. Matrices prepared from pieces ofmaterial smaller than the above range have small pore sizes and completeremoval of cells is difficult; and larger pieces present a smallersurface area.

A nutrient medium used in the method of this invention must containsources of readily assimilable carbon, nitrogen, and oxygen. The mediumis buffered to the correct physiological pH range (6.5-8.0) and may besupplemented with metal salts such as Earle's salts, ascorbic acid,nonessential amino acids such as; alanine, asparagine, aspartic acid,glycine, glutamic acid, proline and serine, or a serum such as foetalbovine serum. Where necessary the medium may also contain antibioticssuch as penicillin. When culturing mammalian cells, any medium normallyemployed for such cells may be used in this method. Suitable mediainclude: Eagle's, Fischer's, Ham's, Leibovitz, McCoy's, Neumann andTytell's, Puck's Swim's, Trowell's or Waymouth's medium, also 199, NCTC109, NCTC 135, CMRL 1066, or RPML medium.

The foam may be generated in the medium by any standard method of foamgeneration, for example, by beating gas into the medium with one or moremechanical whisks. Alternatively the foam may be generated relying ongas dissolved in the medium. By using a cavitating pump in which animpeller rotates at high speed, areas of low pressure are set up in theliquid medium in which bubbles form thereby causing a foam. In yetanother method, the gas may be introduced into liquid by a single ormultiple, static or moving, gas inlets or with a sintered glass bulb.

The foam may be generated from any non-toxic gas or mixture of gases.The foam may be generated in the medium using a completely inert gas,and the aerobic conditions required for culturing the animal cells maybe generated by subsequently introducing the required amount of oxygeninto the system. We have also found that by introducing carbon dioxideinto the culture system, an equilibrium is set up between the carbondioxide in the gas phase and in the liquid phase. This equilibriumprovides a buffering effect which assists in maintaining a uniform pHthroughout the culture system. When the foam is first generated in theculture medium with an inert gas and then supplemented with a secondgas, it is preferable that the second gas be introduced to the culturesystem at a point beyond which the foam has come into contact with thematrix and broken down, so as to prevent any deterioration of the foam.Alternatively, the foam may be generated directly using a gas requiredmixture containing oxygen and optionally carbon dioxide. In suchcircumstances an inert diluent gas must be included to prevent oxidizingconditions being set up within the culture which would be detrimental tothe cells. Similarly where carbon dioxide is included we have found thatthe gas mixture preferably does not contain oxygen and carbon dioxidealone, since toxic effects are often observed and thus a diluent such asnitrogen should be added.

For the aerobic culture of mammalian cells (e.g. fibroblasts) mixturescontaining from 1 to 15% v/v of oxygen and 99 to 85% nitrogen aresuitable and a preferred mixture is one containing 10% oxygen and 90%nitrogen. In the case of foams for aerobic cultures generated from gasmixture also containing carbon dioxide suitable proportions are 7 to 10%v/v of oxygen 0.5 to 7.5% v/v of carbon dioxide made up to 100% withnitrogen. One preferred mixture contains 5% carbon dioxide, 10% oxygenand 85% nitrogen.

Of course the foam could be generated from a gas neither containing norbeing supplemented with carbon dioxide and that the pH of the mediumcould be controlled by standard buffer system.

The foam may be stabilized by supplementing the medium with a non-toxicsurfactant. Examples of surfactants which are useful in this way aresera such as foetal bovine serum; bactopeptone, lipoic acid, linoleicacid, methylcellulose, carboxymethylcellulose, PLURONIC F68 and TWEEN80. [PLURONIC and TWEEN are Registered Trade Marks]. Some media, forexample, McCoy's medium, Ham's medium and media NCTC 109, NCTC 135, CMRL1066 already contain surfactants such as bactopeptone, lipoic acid,linoleic acid and Tween 80. We have found that although these media canbe made to foam and flow as a thin film, additional surfactant mayadvantageously be added in order to obtain satisfactory culturing. Theadditional surfactant employed may be a further amount of the surfactantalready present in the medium or any of the different surfactantspreviously noted.

When the foam has been generated, it is supplied to the solid matrix. Inorder to achieve this supply, the foam generator is situated so as tosupply foam to at least part of the matrix. The transference of the foamfrom the generator to the matrix is achieved either by pumping the foamby using for example a peristaltic pump, or making use of the flowimparted to the foam by the generator, e.g., where the foam is generatedin a cavitating pump or by the injection of gas. Alternatively the pointof generation of the foam may be situated above the point of applicationso that the foam may flow to the matrix through ducts, under theinfluence of gravity.

A preferred characteristic required of a foam used in the method of thisinvention is that it should be sufficiently stable to arrive at thematrix without having undergone any substantial collapse. Foams of lowstability which collapse between the point of generation and applicationtend to set up a liquid flow which submerges the matrix and theadvantages of the film culture are not achieved. This undesired liquidflow may be drained from the foam for recycling or run to waste beforeit arrives at the matrix, but this tends to reduce the efficient use ofmedium.

The characteristics of any particular foam will be determined by theproportion of surfactant employed, and the size of the gas bubblesintroduced into the liquid. The stability of the foam is enhanced byincreasing the proportion of the surfactant and decreasing the size ofthe gas bubbles. The particular foam of choice will depend upon thedistance the foam must travel from the generator to the point ofapplication. These features will in turn depend upon the particularapparatus employed for carrying out the method of this invention, butmay quite easily be determined by generating a foam and observing itsdegree of collapse in transfer. We have found that a foam generatedusing a nutrient medium containing at least 0.05% additional surfactantin which gas bubbles have mean diameters within the range 0.1 to 2mminclusive may travel through a distance of at least 175cm without anysubstantial collapse of foam. A preferred foam for use under theseparticular circumstances contains 0.1% surfactant or 5% or 10% of serumand bubbles of substantially 0.5mm mean diameter.

The rate at which the foam is supplied to the matrix should besufficient to contact substantially all the cell monolayer on thematrix. The rate at which the foam is supplied is dependent upon thetype of matrix employed and the cross-sectional area which it presentsto the foam. We have found that a suitable rate of flow of foam tosupply to a matrix made up of spheres having diameters in the range2-4mm or cylinders having diameters of 1-10mm and lengths of 1-10mm, isbetween 20 and 320cm³ hr⁻¹ per cm² of bed cross-sectional area.

The cells may be washed after culturing and before removal with anysuitable physiological buffer solution supplemented by surfactant, e.g.phosphate buffered brine supplemented with methylcellulose.

The method of this invention is particularly useful for the preparationof cell products such as interferon, or for the production of virusantigens suitable for inclusion in vaccines.

Thus the cells which are supported on the matrix in a monolayer, priorto being contacted with a film of liquid nutrient medium according tothe invention, may be treated with an interferon inducer or with aninfective virus. Such a treatment of the cells may be performed eitherbefore or after the cells are supported on the matrix. The treatment mayalso advantageously be performed using a film derived from the breakdownof a foam.

Thus the invention also provides a method for the treatment of cellswhich method comprises contacting a monolayer of mammalian cellssupported on the surface of a solid porous matrix with a liquid mediumcontaining either an interferon inducer or an infective virus, theliquid medium being in the form of a film derived from the breakdown ofa foam generated from liquid medium and a gas.

Interferon may be induced in such cells on incubation with inducingsubstances such as viruses, virus particles, double stranded ribonucleicacid (ds RNA) of viral origin and synthetic double stranded ribonucleicacids (ds RNA). Double stranded ribonucleic acids having this capacitymay be isolated from viruses infecting certain fungi such as Penicilliaand Aspergilli as described in, for example, British Patent No. 1170929,Banks et. al. Nature 218 542 (1968) and in our British Patent No.1300259.

Most suitably the interferon inducer employed in the method of thisinvention is a double stranded ribonucleic acid of viral origin, asfound in virus particles infecting Penicillia, for example, P.chrysogenum (British Pat. No. 1170929); P. stoloniferum (Banks et. al.,Nature 218 542 (1968)); P. cyaneofulvum (Banks et. al., Nature 213 155(1968)) and in Aspergilli, for example, A. niger and A. foetidus(British Pat. No. 1300259). Preferably the ds RNA is as found in viralparticles which infect P. chrysogenum (British Pat. No. 1170929).

For viral infection, the particular species from which the cell isobtained depends on the type of vaccine which is to be prepared. Virusantigens for incorporation into pharmaceutical vaccines are producedfrom the cells of higher primates such as monkey or man but arepreferably obtained from human cells. Virus antigens for incorporationinto veterinary vaccines are produced from a suitable animal species.

The cells are infected by incubating the cells with the infective virus.Examples of viruses which may be produced by this method for thepreparation of pharmaceutical vaccines include Rabies, Measles, Mumps,German Measles, Polio, myelitis, Adenovirus, Yellow fever,Parainfluenza, Herpes, Cytomegalovirus, Influenza, ParainfluenzaHepatitis, Infectious mononucleosis and Respiratory syncytial virus.

Examples of viruses which may be produced for the preparation ofveterinary vaccines include Mareks disease, Parainfluenza, Adenovirus,Distemper, Cat leukaemia, Infectious bovine rhinotracheitis, Infectiousbronchitis, Foot and Mouth Disease, Calf diarrhoea, Equinerhinopheumonitis, Pseudorabies, Infectious laryngotracheitis, Swinevesicular, Transmiscible gastroenteritis.

The types of cells which are suitable for use in this method are thoseanimal cells which are capable of growing in sheets, for example,fibroblasts and epithelial cells as previously discussed. Forconvenience skin and muscle fibroblasts are preferred.

Prior to induction of infection treatment, the cells are attached to atleast part of the matrix and optionally grown to a confluent sheet bythe method previously described. For this purpose any of the mediapreviously discussed are suitable. Where fibroblasts are used, aparticular medium is Eagle's medium supplemented with bovine foetalserum, bovine brain extract, non-essential amino acids, Earle's salts,ascorbic acid and an antibiotic and buffered with sodium bicarbonate.

For interferon induction, interferon is induced in the cell sheets byeither submerging the matrix in an induction medium or introducing theinduction medium as a thin film derived from the breakdown of a foamgenerated in an induction medium supplemented with a surfactant.Suitably this induction incubation is performed at a temperature withinthe range generally suitable for cell growth, i.e. 35° to 38° C.preferably about 37° C. This induction incubation may be carried out fora period of between one and three hours. We have found that two hours isa convenient period. The induction medium is a solution comprising theds RNA in any of the conventional culture media optionally supplementedby any of the surfactants previously discussed. It is generally mostconvenient to dissolve the ds RNA in the same medium as employed for thecell culture. Where fibroblast cells are used, a preferred inductionmedium is one in which the ds RNA is dissolved in minimum essential(Eagle's) medium supplemented with an antibiotic and DEAE dextran andbuffered to a physiological pH with sodium bicarbonate.

For viral infection, the cells are then infected by either submergingthe matrix with an infection medium or introducing the infection mediumas a thin film derived from the breakdown of a foam generated in aninfection medium supplemented with a surfactant. The infection medium isa solution comprising the virus in any of the conventional culture mediaoptionally supplemented with any of the surfactants previouslydiscussed. It is generally most convenient to suspend the virus in thesame medium as employed for the cell culture. Where fibroblast cells areused, a preferred infection medium is one in which the virus issuspended in minimum essential (Eagle's) medium supplemented with anantibiotic and DEAE dextran and buffered to a physiological pH withsodium bicarbonate. Suitably this infection incubation is performed at atemperature within the range generally suitable for cell growth, i.e.35° to 38° C. preferably about 37°. The infection incubation may becarried out for a period of between one and three hours. We have foundthat two hours is a convenient period.

After the induction of infection period, the cells are washed free ofinfection medium with phosphate buffered saline either by immersion orby supplying the saline as a thin film and a collection medium issupplied to the cell sheet as a thin film for a period of between 6-25hours, preferably 16-20 hours for the collection of interferon andbetween 3 to 7 days for the collection of virus. The collection mediummay be any of the typical culture media as previously discussed. It isgenerally most convenient to use the same medium as used for cellculture, in the case of fibroblast cells, particularly suitablecollection medium is minimum essential (Eagle's ) medium andsupplemented with foetal bovine serum, and an antibiotic and bufferedwith sodium bicarbonate to a physiological pH. The collection is carriedout in the temperature range generally employed for the culture ofcells, i.e. 35° to 38° C. preferably 37° C. After the collection period,the circulation of the collection medium is stopped, the matrix drainedand medium containing interferon or virus is then collected. Theinterferon or virus content of the solution may then be assayed and theinterferon or virus isolated by any of the standard methods.

In another of its aspects this invention provides apparatus in whichthis method may be performed.

Accordingly this invention provides apparatus comprising a vesselcontaining a solid porous matrix which permits the adherence of cells, afoam generator located to supply foam to at least part of the surface ofthe solid porous matrix and means for removing liquid from the vessel.

The vessel suitable for use in this invention may be a tower or troughof circular or rectangular cross-section, or in the shape of a hollowcone, invert cone or sphere. The vessel will have an inlet through whichthe matrix can be introduced, and an outlet from the foam generatprthrough which the foam is supplied to the matrix. The vessel may also beprovided with a further inlet for introducing the suspension of cells.This may take the form of a self-sealing rubber diaphragm or a standardsteam sterilizable inlet port. In order to ensure that thecharacteristics of the medium remain in the desired range, the vesselmay be provided with pH and/or pO₂ sensitive electrodes.

The vessel may also be provided with a gas outlet optionally through asterile filter to allow the escape of gas from the foam. The vessel mayalso be provided with an additional gas inlet positioned suitably beyondthe point at which the foam collapses onto the matrix so that the influxof gas does not break up the foam. Medium is supplied to the foamgenerator from a reservoir suitably by gravity feed, or optionally bypumps.

The efflux of medium from the vessel may be run to waste or return bypumping where necessary to the medium reservoir.

Apparatus in accordance with this invention will now be described by wayof example with reference to the accompanying FIG. 1, which is aschematic diagram of apparatus according to this invention.

Referring to FIG. 1, a cell culture apparatus in accordance with thisinvention may be seen to comprise, a foam generator, 1; a vessel in theform of a cylindrical tower, 2; containing a solid porous matrix, 3; thevessel having an outlet, 4; as means for removing liquid.

In this particular embodiment the foam generator, 1; consists of acylindrical housing, 5; a gas inlet pipe, 6; which is attached at oneend to a flow meter, 6a; and which towards the other end passes into thehousing through the side wall, and terminates in a sintered glass disc,7. The generator, 1; also has a liquid inlet, 8; at one end of thehousing and a foam outlet, 9; at the other end of the housing. Theinlet, 8; is connected to a liquid reservoir, 10; via a supply pipe, 8a;having a non-return valve, 11. The liquid supply pipe in this embodimentmay be drained by valve, 12; situated between the non-return valve andthe foam generator. The foam outlet, 9; is connected to one end of afoam supply pipe, 9a; which terminates within the vessel, 2; above thetop surface, 13; of the solid porous matrix, 3.

In this embodiment the tower, 2; contains a matrix, 3; of small plasticcylinders. The tower is closed at the top end by a tightly fitting boredbung, 14; through which the foam supply pipe, 9a; passes. The towertapers at the lower end to form an outlet, 4. The tower, 2; also has aninlet for cell suspensions, in the form of a self-sealing rubberdiaphragm, 15; and a gas outlet pipe, 16; terminating in a bacterialfilter, 17. The outlet, 4; is connected to one end of a withdrawal pipe,18. The withdrawal pipe, 18; is divided into two parts, one part, 18a;of which returns via a valve, 19; to the liquid reservoir, 10; and theother part, 18b; forms a drainage outlet through the valve, 20. Thereservoir, 10; to which medium is returned has a gas outlet pipe, 22;and a liquid inlet in the form of a self-sealing rubber diaphragm, 23.The gas outlet, 22; in this embodiment joins the gas outlet pipe, 16;from the vessel, 2; below the bacterial filter, 17.

In order to use the device, the tower, 2; is filled with a convenientquantity of polycarbonate cylinders, the bored bung, 14; is insertedinto the tower and the apparatus assembled. The entire apparatus is thensterilized. The apparatus is then placed in a constant temperatureenvironment (ca 37° C.) and allowed to come to ambient temperature.Cells suspended in sufficient medium to immerse the matrix areintroduced to the tower, 2; via the self-sealing diaphragm, 15; and theapparatus is allowed to stand for between 2 and 20 hours to allow thecells to settle onto the matrix. Nutrient medium is introduced to thereservoir, 10; through the self-sealing rubber diaphragm, 23; and isallowed to flow into the foam generator, 1; via pipe 8a. A sterile gasis introduced into the generator at a rate metered by flowmeter, 6a;which causes the medium to foam. The foam is swept along the pipe, 9; tothe top of the matrix by the gas flow. As the foam arrives at the matrixit breaks down and the liquid medium flows over the matrix surface. Atthe same time the valve, 20; is opened to allow the efflux of thesuspension medium at a rate sufficient to allow its replacement bynutrient medium derived from the foam, and to prevent the cell layerbecoming dry. When the suspension medium has been replaced by nutrientmedium derived from the foam, the drainage valve, 20; is closed and thevalve, 19; is opened so that the medium returns to the reservoir, 10.The nutrient medium is recycled until a colony of cells of the requiredsize has been established. The gas supply is then shut off, and thevalve, 19; closed. The reservoir, 10; and the foam generator are drainedof nutrient medium and filled with wash medium. The gas supply isrestarted. The cells are washed with a thin film of wash medium untilfree of culture medium. The effluent is either allowed to run to wastethrough the valve, 20; or is recycled to the reservoir, 10; through thevalve, 19. The supply of foam is then stopped and the cells are removedfrom the matrix suitably by filling the culture vessel with a cellrelease solution such as trypsin. The matrix may be optionally agitatedgently to assist in freeing the cells by slowly passing gas through thematrix from below through the pipe, 18; while keeping valve 19 closed.

The suspension of cells is then withdrawn through the drainage valve,20; while keeping valve 19 closed and valve 20 open.

The apparatus may be provided optionally with a vapour trappingcondenser below the bacterial filter, 17; and an optional additional gassupply situated so as to introduce gas at a point below which thewithdrawal pipe, 18; divides into the two parts 18a and 18b.

The invention is illustrated by the following Examples.

EXAMPLE 1

The apparatus as described in specification having a vessel 2 of 3.5 cmdiameter was filled to a depth of 25 cm with moulded poly-carbonatecylinders 4 mm in diameter and 4 mm long. The entire apparatus wassterilized by autoclaving at 15 psi for 30 minutes.

The apparatus was then allowed to cool to room temperature and withvalves 19 and 20 closed an inoculum of 3.6 × 10²⁷ L929 mouse fibroblastcells suspended in 100 cm³ of growth medium were introduced via theinlet point 15 using a hypodermic syringe. The growth medium used wasEagle's medium, supplemented with Earle's salts, 10% Foetal bovineserum, non-essential amino acids, ascorbic acid andpenicillin/streptomycin. The medium was buffered with 2.2 g of sodiumbicarbonate per liter and contained the pH indicator, phenol red.

The inoculated apparatus was allowed to stand for 20 hours at 37° inorder to let the cells settle and attach themselves to the packing.

When the cells had settled on to the porous matrix the valve 19 wasopened slightly to allow the liquid to drain from the vessel and at thesame time a gas mixture containing 10% oxygen + 85% nitrogen + 5%carbondioxide, was supplied to the foam generator at a rate of 25 cc/minso that foam arrived at the top surface of the porous matrix as theliquid present drained away. The rate of flow of medium through valve 19was controlled so that the medium in which the cells had been suspendedis replaced by medium derived from the foam and the cells on the matrixdid not become dry. When the medium in which the cells had beensuspended was completely drained from the tower the valve 19 wascompletely opened and the rate of flow of liquid through the column wascontrolled by the rate of breakdown of foam at the surface of thematrix.

The foam was allowed to circulate for 72 hours.

The supply of gas to the generator 1 was stopped and valve 19 closed.The reservoir 10 and foam generator 1 were drained through valve 12.With valve 12 closed the reservoir 10 was filled with a wash medium 0.15molar brine buffered to pH 7.3 with phosphate and supplement with 0.1%methyl cellulose. The wash medium was caused to foam and pass throughthe vessel 2 and run to waste through valve 20 via pipe 18b, until thecells were free from the last traces of the red nutrient medium. Thevalve 20 was closed and trypsin/versene solution at 37° C. wasintroduced into the vessel via the inlet 15 to immerse the porousmatrix, and after 10 minutes the packing the solution was gentlyagitated for five minutes by introducing via valve 20, a slow gentlestream of sterile air. The cells having been suspended in the buffer asa result of the agitation are drained from the vessel 2, via valve 20. Afurther portion of trypsin/versene buffer was added to remove anyremaining cells.

The number of cells harvested was determined by counting them using ahaemocytometer.

    ______________________________________                                        Total cells                        3.6 × 10.sup.7                        inoculated                                                                   Cells harvested                                                                          (i)   in drained growth medium                                                                        0.8 × 10.sup.7                                        and saline wash buffer                                                  (ii)  in Trypsin/versene                                                                              5.25 × 10.sup.7                      Total                              6.05 × 10.sup.7                      ______________________________________                                    

EXAMPLE 2

Using the method of example 1 human fibroblasts were cultured andinterferon produced as follows:

A 5 cm × 15 cm matrix of 4 mm silicone treated polycarbonate beadspacked into a cylindrical glass tower was inoculated with 2.5 × 10⁷human skin/muscle fibroblasts. The cells were added as a suspension ingrowth medium comprising minimum essential medium (Eagle) with Earle'ssalts supplemented with 10% foetal bovine serum, non-essential aminoacids, ascorbic acid, fibroblast growth factor. penicillin/streptomycinand 0.88 g/l sodium bicarbonate.

The inoculated matrix was incubated and left to stand overnight at 37°C. The suspension medium was slowly drained and incubation with nutrientcommenced by supplying a mixture of 5% carbon dioxide in sterile air toliquid in the foam generator. This mixture was adjusted to give thecorrect pH level (7.0-7.2) in the medium. The medium circulation wascontinued for 48 hours.

Medium was allowed to accumulate in the matrix which was then washed byoverlaying the beads and medium and slowly draining liquid from the baseof the matrix. Induction medium was added in a similar manner andcomprised minimum essential (Eagle) medium (MEM) supplemented withEarle's salts, sodium bicarbonate, penicillin/streptomycin, 5 ug/ml dsRNA and 100 ug/ml DEAE dextran. This was left in contact with the cellsfor 2 hours at 37° C. and then washed off with PBS as before. The PBSwash was displaced by collection medium (MEM, sodium bicarbonate,penicillin/streptomycin and 1% foetal bovine serum). Tower leftovernight at 37° C. in this condition when medium was drained off andfound to contain human interferon (3,300 international units).

EXAMPLE 3

Using the method of example 1, human fibroblasts were cultured asfollows:

A 3.5 cm × 25 cm matrix of 4 mm polycarbonate beads, which had beensprayed with PTFE aerosol spray (Fisons) and packed into a cylindricalglass tower, were inoculated with 8 × 10⁶ human skin/muscle fibroblasts.The cells were added as a suspension in growth medium comprising minimumessential medium (Eagle's) with Earle's salts supplemented with 10%foetal bovine serum, non-essential amino acids, penicillin/streptomycinand 0.88 g/l sodium bicarbonate.

The inoculated matrix was incubated at 37° C. overnight. The suspensionmedium was slowly drained and circulation of nutrient commenced bysupplying a mixture of air and carbon dioxide to liquid in the foamgenerator. This mixture was adjusted to give the correct pH level(7.0-7.2) in the medium. The medium circulation was continued for 75hours.

The supply of gas to the generator 1 was stopped and valve 19 closed.The reservoir 10 and foam generator 1 were drained through valve 12.With valve 12 closed the reservoir 10 was filled with a wash medium 0.15molar brine buffered to pH 7.3 with phosphate and supplemented with 0.1%methyl cellulose. The wash medium was caused to foam and pass throughthe vessel 2 and run to waste through valve 20 via pipe 18b, until thecells were free from the last traces of the red nutrient medium. Thevalve 20 was closed and trypsin/versene solution at 37° C. wasintroduced into the vessel via the inlet 15 to immerse the porousmatrix, and after 10 minutes the packing and solution was gentlyagitated for 5 minutes by introducing via valve 20 a slow stream ofsterile air. The cells having been suspended in the buffer as a resultof the agitation are drained from the vessel 2, via valve 20. Theagitation was repeated using 0.15 M brine buffered to pH 7.3 withphosphate. A further portion of trypsin/versene buffer was addedfollowed by a further portion of brine both of which were agitated toremove any remaining cells.

    ______________________________________                                        Total cells                        8 × 10.sup.6                          inoculated                                                                   Cells harvested                                                                          (i)    in drained growth medium                                                                       0.5 × 10.sup.6                                         and saline wash buffer                                                 (ii)   in 1st trypsin/versene                                                                         10.7 × 10.sup.6                                        treatment                                                              (iii)  in 1st agitated saline                                                                         1.95 × 10.sup.6                                        wash                                                                   (iv)   in 2nd trypsin/versene                                                                         2.48 × 10.sup.6                                        treatment                                                              (v)    in 2nd agitated saline                                                                         4.13 × 10.sup.6                                        wash                                                                          Total            19.8 × 10.sup.6                      ______________________________________                                    

In the following table examples are given of the method of the inventionemployed to culture various human fibroblasts using differing matrices.

The abbreviations used therein have the following meanings:

1000: Flow 1000 human skin and muscle fibroblasts

7000: Flow 7000 human foreskin fibroblasts

12000: Flow 12000 human epithelial nasal mucosal cells

meth: methylcellulose

F.g.f.: fibroblast growth factor

P.c.: poly carbonate

Vit.c.: ascorbic acid

N 6: nylon 6

N 11: nylon 11

N 12: nylon 12

Pet: polyethylene terephthalate

Pet(o): oxygenated polyethylene terephthalate

Ppo: polyphenyleneoxide

Pp: polypropylene

Pe: polyethylene

Pf: polyformaldehyde

Fep: fluorinated ethylene propylene co-polymer

P.t.f.e.: poly tetrafluoroethylene

S: silicone

Sil: silicone

D.c. sil: Dow Corning Silicone 1107

Stearic: Stearic acid

A: air

N: nitrogen

O/n: overnight

Inoculation and Harvest are measured in: Number of cells × 10⁷

    __________________________________________________________________________                                                     Growth    Attach-                                                             period    ment               Example                                                                            Cells Serum                                                                             Additives                                                                             Packing Gas   Inoculation                                                                          Harvest                                                                            in days                                                                             Ratio                                                                             Period             __________________________________________________________________________     4   1000(P25)                                                                           10% --      PC      A/N/CO.sub.2                                                                        ≦2.5                                                                          1.78 4     --  O/N                 5   1000(P25)                                                                           10% 0.1% meth                                                                             PC      A/N/CO.sub.2                                                                        2.5    2.07 4     --  O/N                 6   1000(P26)                                                                           10% --      PC      A/N/CO.sub.2                                                                        1.35   1.15 4     --  O/N                 7   1000(P26)                                                                           10% --      PC      A/N/CO.sub.2                                                                        1.35   1.26 4     --  O/N                 8   1000(P28)                                                                           10% --      PC      A/N/CO.sub.2                                                                        1.7    1.35 8     --  O/N                 9   1000(P28)                                                                           10% --      PC      A/N/CO.sub.2                                                                        1.7    1.71 11    1   O/N                10   1000(P29)                                                                           10% --      N 11    A/N/CO.sub.2                                                                        1.9    1.46 8     --  O/N                11   1000(P29)                                                                           10% --      PET(O)  A/N/CO.sub.2                                                                        1.6    1.61 8     1   O/N                12   1000(P29)                                                                           10% --      PC/S    A/N/CO.sub.2                                                                        1.6    1.72 8     1.08                                                                              O/N                13   1000(P29)                                                                           10% --      PC/PTFE A/N/CO.sub.2                                                                        1.6    3.05 8     1.9 O/N                14   7000(P24)                                                                           10% --      GLASS   A/N/CO.sub.2                                                                        1.5    0.72 9     --  O/N                15   7000(P24)                                                                           10% --      GLASS/S A/N/CO.sub.2                                                                        1.5    0.74 9     --  O/N                16   7000(P24)                                                                           10% --      GLASS/PTFE                                                                            A/N/CO.sub.2                                                                        1.5    0.59 9     --  O/N                17   7000(P25)                                                                           10% --      PC/PTFE A/N/CO.sub.2                                                                        1.4    2.09 8     1.49                                                                              O/N                18   7000(P25)                                                                           10% --      PC/PTFE A/N/CO.sub.2                                                                        1.4    2.9  8     2.07                                                                              O/N                19   7000(P26)                                                                           10% --      PC/PTFE A/N/CO.sub.2                                                                        1.8    1.92 4     1.07                                                                              O/N                20   7000(P26)                                                                           10% --      PC/PTFE A/N/CO.sub.2                                                                        1.8    2.51 6     1.4 O/N                21   7000(P26)                                                                           10% --      PC/PTFE A/N/CO.sub.2                                                                        1.8    2.80 5     1.56                                                                              O/N                22   7000(P26)                                                                           10% --      PC/PTFE A/N/CO.sub.2                                                                        1.8    5.18 11    2.88                                                                              O/N                23   7000(P26)                                                                           10% --      PC/PTFE A/N/CO.sub.2                                                                        1.8    3.64 7     2.02                                                                              O/N                24   7000(P27)                                                                           10% --      PC/PTFE A/CO.sub.2                                                                          1.05   2.94 4     2.8 O/N                25   7000(P27)                                                                           10% --      PC/PTFE A/CO.sub.2                                                                          1.25   2.95 4     2.36                                                                              O/N                26   1000(P37)                                                                           10% --      PC/PTFE A/CO.sub.2                                                                          0.9    0.99 1     1.10                                                                              O/N                27   1000(P37)                                                                           10% --      PC/PTFE A/CO.sub.2                                                                          0.9    1.18 2     1.31                                                                              O/N                28   1000(P37)                                                                           10% --      PC/PTFE A/CO.sub.2                                                                          0.9    2.27 5     2.52                                                                              O/N                29   1000(P37)                                                                           10% --      PC/PTFE A/CO.sub.2                                                                          0.9    2.11 8     2.34                                                                              O/N                30   1000(P37)                                                                           10% --      PC/PTFE A/CO.sub.2                                                                          0.9    2.47 9     2.75                                                                              O/N                31   1000(P39)                                                                           10% --      PC/PTFE A/CO.sub.2                                                                          0.8    1.98 4     2.47                                                                              O/N                32   1000(P39)                                                                           10% --      PC/PTFE A/CO.sub.2                                                                          0.8    2.13 4     2.66                                                                              O/N                33   1000(P40)                                                                           10% --      N6/PTFE A/CO.sub.2                                                                          0.6    0.75 4     1.25                                                                              O/N                34   1000(P40)                                                                           10% --      N11/PTFE                                                                              A/CO.sub.2                                                                          0.6    uncount-                                                                           4     --  O/N                                                            able                              35   1000(P40)                                                                           10% --      N12/PTFE                                                                              A/CO.sub.2                                                                          0.6    uncount-                                                                           4     --  O/N                                                            able                              36   1000(P40)                                                                           10% --      PC/PTFE A/CO.sub.2                                                                          0.6    0.94 4     1.57                                                                              O/N                37   7000(P32)                                                                           10% --      PF      A/CO.sub.2                                                                          1.0    1.3  4     1.3 O/N                38   7000(P32)                                                                           10% --      PF/PTFE A/CO.sub.2                                                                          1.0    0.335                                                                              4     --  O/N                39   7000(P32)                                                                           10% --      PC/PTFE A/CO.sub.2                                                                          1.0    1.07 7     1.07                                                                              O/N                40   7000(P31)                                                                           10% --      PET/PTFE                                                                              A/CO.sub.2                                                                          0.8    0.96 4     1.20                                                                              O/N                41   7000(P31)                                                                           10% --      PET(O)/PTFE                                                                           A/CO.sub.2                                                                          0.8    0.88 4     1.10                                                                              O/N                42   7000(P31)                                                                           10% --      PC/PTFE A/CO.sub.2                                                                          0.8    contami-                                                                           --    --  O/N                                                            nated                             43   1000(P23)                                                                           10% --      PET/PTFE                                                                              A/CO.sub.2                                                                          1.25   0.89 4     --  O/N                44   1000(P23)                                                                           10% --      PET(O)/PTFE                                                                           A/CO.sub.2                                                                          1.25   1.27 4     1.02                                                                              O/N                45   1000(P23)                                                                           10% --      PC/PTFE A/CO.sub.2                                                                          1.25   1.26 4     1.01                                                                              O/N                46   1000(P24)                                                                           10% --      PET/PTFE                                                                              A/CO.sub.2                                                                          1.375  induced                                                                            7     --  O/N                47   1000(P24)                                                                           10% --      PET(O)/PTFE                                                                           A/CO.sub.2                                                                          1.375  induced                                                                            8     --  O/N                48   1000(P24)                                                                           10% --      PC/PTFE A/CO.sub.2                                                                          1.375  induced                                                                            9     --  O/N                49   1000(P26)                                                                           10% --      FEP     A/CO.sub.2                                                                          1.0    0.25 4     --  O/N                50   1000(P26)                                                                           10% --      PPO     A/CO.sub.2                                                                          1.0    0.685                                                                              4     --  O/N                51   1000(P26)                                                                           10% --      PPO/PTFE                                                                              A/CO.sub.2                                                                          1.0    0.91 4     --  O/N                52   1000(P26)                                                                           10% --      PC/PTFE A/CO.sub.2                                                                          0.725  induced                                                                            9     --  O/N                53   1000(P26)                                                                           10% --      PC/PTFE A/CO.sub.2                                                                          0.725  induced                                                                            8     --  O/N                54   1000(P26)                                                                           10% --      PC/PTFE A/CO.sub.2                                                                          0.725  induced                                                                            10    --  O/N                55   12000(P23)                                                                          10% --      PC/PTFE A/CO.sub.2                                                                          1.2    1.24 4     1.03                                                                              O/N                56   12000(P23)                                                                          10% --      FEP     A/CO.sub.2                                                                          1.2    0.41 4     --  O/N                57   12000(P23)                                                                          0   0.1% Meth                                                                             FEP     A/CO.sub.2                                                                          0.8    0.59 4     --  O/N                58   12000(P24)                                                                           5% 5% FGF  PC/PTFE A/CO.sub.2                                                                          0.95   0.77 3     --  O/N                59   12000(P24)                                                                          10% --      PC/PTFE A/CO.sub.2                                                                          1.05   0.79 3     --  O/N                60   12000(P24)                                                                          10% --      PC/PTFE A/CO.sub.2                                                                          1.05   1.18 3     1.12                                                                              O/N                61   12000(P25)                                                                           5% --      PC/PTFE A/CO.sub.2                                                                          0.8    0.675                                                                              4     --  21/2hr.            62   12000(P25)                                                                           5% --      PC/PTFE A/CO.sub.2                                                                          0.8    0.503                                                                              4     --  16hr.              63   12000(P25)                                                                           5% --      PC/PTFE A/CO.sub.2                                                                          0.8    0.734                                                                              4     --  16hr.              64   1000(P30)                                                                            5% --      PC/PTFE A/CO.sub.2                                                                          1.0    2.03 4     2.03                                                                              21/4hr.            65   1000(P30)                                                                            5% --      PC/PTFE A/CO.sub.2                                                                          1.0    1.45 4     1.45                                                                              21/4hr.            66   1000(P24)                                                                           10% --      PC/PTFE A/CO.sub.2                                                                          1.375  1.23 4     --  O/N                67   1000(P24)                                                                           10% --      PC/PTFE A/CO.sub.2                                                                          1.375  1.175                                                                              4     --  O/N                68   1000(P25)                                                                            5% --      PC/PTFE A/CO.sub.2                                                                          0.925  0.57 4     --  3hr.               69   1000(P25)                                                                            5% --      PC/PTFE A/CO.sub.2                                                                          0.925  0.42 4     --  5hr.               70   1000(P25)                                                                            5% --      PC/PTFE A/CO.sub.2                                                                          0.925  0.13 4     --  7hr.               71   1000(P25)                                                                            5% --      PC/PTFE A/CO.sub.2                                                                          0.925  0.21 4     --  9hr.               72   1000(P27)                                                                           10% --      PP      A/CO.sub.2                                                                          0.72   0.34 4     --  3hr.               73   1000(P27)                                                                           10% --      PP      A/CO.sub.2                                                                          0.72   0.39 4     --  5hr.               74   1000(P28)                                                                            5% FGF     PP      A/CO.sub.2                                                                          1.04   0.41 4     --  3hr.               75   1000(P28)                                                                            5% FGF     PP      A/CO.sub.2                                                                          1.04   0.53 4     --  3hr.               76   1000(P28)                                                                            5% FGF     PP      A/CO.sub.2                                                                          1.3    0.86 4     --  3hr.               77   1000(P29)                                                                            5% FGF     PC      A/CO.sub.2                                                                          1.2    0.91 4     --  3hr.                              +                                                              78   1000(P29)                                                                            5% VITC    PC      A/CO.sub.2                                                                          1.2    0.53 4     --  3hr.               79   12000(P25)                                                                           5% --      PC      A/CO.sub.2                                                                          1.7    induced                                                                            3     --  3hr.               80   12000(P26)                                                                           5% VITC    PC/Sil  A/CO.sub.2                                                                          1.9    1.40 4     --  3hr.               81   12000(P26)                                                                           5% VITC    PC/Sil  A/CO.sub.2                                                                          1.9    1.46 4     --  3hr.               82   1000(P26)                                                                            5% FGF/VITC                                                                              PC/Sil  A/CO.sub.2                                                                          2.5    induced                                                                            2     --  3hr.               83   1000(P29)                                                                           10% --      PC/DC.Sil                                                                             A/CO.sub.2                                                                          2.1    1.32 4     --  --                 84   1000(P29)                                                                           10% --      PC/Sil  A/CO.sub.2                                                                          2.1    1.29 4     --  --                 85   1000(P29)                                                                           10% --      PC/PTFE/Sil                                                                           A/CO.sub.2                                                                          2.1    1.62 4     --  --                 86   1000(P31)                                                                            5% --      PC/Sil  A/CO.sub.2                                                                          1.7    0.7  4     --  3hr.               87   1000(P31)                                                                            5% --      GLASS/Stearic                                                                         A/CO.sub.2                                                                          1.7    0.6  4     --  3hr.               88   1000(P33)                                                                            5% FGF     PC/Sil  A/CO.sub.2                                                                          1.7    1.13 4     --  3hr.               89   1000(P33)                                                                            5% FGF     PC/SIl  A/CO.sub.2                                                                          1.7    0.9  4     --  3hr.               90   1000(p33)                                                                            5% FGF     PC/DC.Sil                                                                             A/CO.sub.2                                                                          1.7    2.2  4     1.3 3hr.               91   12000(P33)                                                                           5% VITC    PP      A/CO.sub.2                                                                          1.7    2.06 4     1.21                                                                              3hr.               92   12000(P33)                                                                           5% VITC    PE      A/CO.sub.2                                                                          1.7    1.67 4     1.0 3hr.               93   12000(P33)                                                                           5% VITC    PP      A/CO.sub.2                                                                          1.7    1.79 4     1.05                                                                              3hr.               94   1000(P35)                                                                            5% FGF/VITC                                                                              PP      A/CO.sub.2                                                                          1.36   1.16 4     --  3hr.               95   1000(P35)                                                                            5% FGF/VITC                                                                              PP      A/CO.sub.2                                                                          1.36   0.58 4     --  3hr.               96   1000(P35)                                                                            5% FGF/VITC                                                                              PE      A/CO.sub.2                                                                          1.36   0.76 4     --  3hr.               97   1000(P23)                                                                            5% FGF/VITC                                                                              PP      A/CO.sub.2                                                                          1.78   0.896                                                                              4     0.54                                                                              3hr.               98   1000(P23)                                                                            5% FGF/VITC                                                                              PP      A/CO.sub.2                                                                          1.97   induced                                                                            2     --  3hr.               99   1000(P23)                                                                            5% FGF/VITC/                                                                             PE      A/N.sub.2                                                                           2.56   1.20 4     0.47                                                                              3hr.                              HEPES                                                          100  1000(P24)                                                                            5% FGF/VITC                                                                              PC/DC.Sil                                                                             A/CO.sub.2                                                                          1.45   2.45 4     1.69                                                                              3hr.               101  1000(P24)                                                                           5%/ FGF/VITC/                                                                             PC/DC.Sil                                                                             A/CO.sub.2                                                                          1.45   1.92 4     1.32                                                                              3hr.                          10% Meth                         (2.38)     (1.64)                 __________________________________________________________________________

What we claim is:
 1. In a method for culturing animal cells in a monolayer supported on the surface of a solid porous matrix the improvement comprising contacting said cells with a film of liquid nutrient medium derived from the breakdown of a foam which is generated from said nutrient medium and a gas leaving gas in interstitial spaces.
 2. A method according to claim 1 wherein said matrix comprises solid granules.
 3. A method according to claim 2 wherein said granules are cylindrical or spherical.
 4. A method according to claim 2 wherein said granules are made of a plastic material, ceramics, glass, an inert metal; or an inert metallic oxide, phosphate, silicate, or carbide.
 5. A method according to claim 1 wherein said cells are mammalian.
 6. A method according to claim 5 wherein said cells are human.
 7. A method according to claim 1 wherein the medium is supplemented with a non-toxic surfactant.
 8. A method according to claim 1 wherein said gas contains up to 10% carbon dioxide.
 9. A method according to claim 1 wherein the foam is sufficiently stable to be transported from its point of generation to its point of application to the matrix without substantial collapse.
 10. A method according to claim 1 wherein said cells are treated with an interferon inducer or an infective virus prior to being contacted with said film of liquid nutrient medium.
 11. In a method for producing interferon in vitro, the improvement which comprises contacting a monolayer of mammalian cells supported on the surface of a solid porous matrix with a liquid medium containing an interferon inducing substance, said liquid medium being in the form of a film derived from the breakdown of a foam generated from said liquid medium and a gas leaving gas in the interstitial spaces; and thereafter separating the interferon.
 12. A method according to claim 11 wherein the said cells are skin and muscle fibroblasts.
 13. A method according to claim 11 wherein the interferon inducing substance is a ds RNA of viral origin.
 14. A method according to claim 13 wherein the ds RNA is isolated from virus particles infecting P. chrysogenum.
 15. In a method for the preparation of a virus in vitro the improvement which comprises contacting a monolayer of animal cells supported on the surface of a solid porous matrix with a liquid medium containing an infective virus, said liquid medium being in the form of a film derived from the breakdown of a foam generated from said liquid medium and a gas leaving gas in the interstitial spaces; and thereafter separating the virus.
 16. A method according to claim 15 wherein the virus is Mareks disease virus or Foot and Mouth disease virus. 